Process for the production of antibiotic X-14547

ABSTRACT

The invention relates to a new and useful antibiotic substance which is of the formula ##STR1## and to processes for its production and recovery. The antibiotic which exhibits ionophoric properties, is classified as a polyether group antibiotic. the antibiotic of formula I is effective in inhibiting the growth of gram positive bacteria and exhibits utility as an antihypertensive agent and as a compound to improve ruminant feed utilization. The antibiotic of Formula I is prepared by cultivating a strain of Streptomyces sp. X-14547 in an aqueous carbohydrate solution containing nitrogenous nutrients and mineral salts and thereafter isolating the antibiotic from the fermentation broth.

This is a division of application Ser. No. 712,286, filed Aug. 6, 1976now U.S. Pat. No. 4,100,171.

DESCRIPTION OF THE INVENTION

There is provided according to the present invention an antibioticsubstance effective in inhibiting the growth of gram-positive bacteriawhich is of the formula ##STR2## wherein Me is methyl and Et is ethyl.

Chemically, this substance is known as α-(R), 5(S)-dimethyl-6(R)-{1-ethyl-4-[4-(R)-(2-pyrrolyl-carbonyl)-1(S)-ethyl-3a(R),4,5(R),7a(R)-tetrahydroindan-5-yl]-1(E),3(E)-butadienyl}tetrahydropyran-2-acetic acid.

There is further provided according to the present invention, afermentation process for the production of such antibiotic substancetogether with the isolation techniques utilized to recover the compoundof Formula I from the fermentation broth.

The organism producing the antibiotic of the present invention is a newspecies designated Streptomyces sp. X-14547. A culture of the livingorganism, given the laboratory designation X-14547 has been deposited inthe U.S. Department of Agriculture, Agriculture Research Service, NRRL,Peoria, Illinois and added to its permanent collection of microorganismsas NRRL 8167. The culture has been identified as a strain ofStreptomyces antibioticus.

The new microorganism was isolated from a soil sample collected atMartinsville, Virginia. The representative strain of Streptomyces sp.X-14547 has the following characteristics:

GENERAL CHARACTERISTICS

Streptomyces X-14547 produces a substrate mycelium which does notfragment into spores, and an aerial mycelium which formsrectus-flexibilis chains of spores; the spores have a smooth surface.The spores are 1.26 to 1.42 μin length and 0.69 to 0.91 μ in width. Thecell wall contains an isomer of diaminopimelic acid other than the mesoform: This fact as well as the above colony characteristics place thisculture in the genus Streptomyces.

GROWTH CHARACTERISTICS

The standard ISP media set forth in Shirling and Gottlieb, "Methods forCharacterization of Streptomyces Species", Intern, J. System.Bacteriol., 16, pp 313-400, 1966, as well as various other media used tocharacterize the culture are listed below:

ISP-1 through ISP-9 are described by Shirling and Gottlieb in abovearticle.

Czapek-Dox: Czapek-Dox Broth (BBL) to which 1.5% agar was added.

Bennett's: Yeast extract, 0.1%, beef extract, 0.1%; N-Z Amine A (caseinhydrolysate from Sheffield, Inc.), 0.2%; dextrose, 1% agar, 1.8% pH 7.3.

Sabouraud Dextrose Agar: (Difco).

Thermoactinomyces Fermentation: Bacto Thermoactinomyces fermentationmedium (Difco) to which 1.5% agar was added.

ATCC 5: Sporulation agar: Yeast extract, 0.1%; beef extract, 0.1%;tryptose, 0.2%; FeSO₄, trace; glucose, 1.0%; agar, 1.5%; pH 7.2.

Amidex: Amidex (Corn Products Co., Decatur, Ill.) 1%; N-Z Amine A, 0.2%;beef extract, 0.1%; yeast extract, 0.1%; CaCl₂.2H₂ O, 0.0014%; agar, 2%;pH 7.3.

Starch casein: soluble starch, 1%; casein, 0.1%; K₂ HPO₄, 0.05%; MgSO₄,0.05%; agar, 1.5%; pH 7.4.

Table 1 below describes the amount of growth, degree of sporulation,spore mass color, and color of the reverse substrate mycelium. Agarplates were read after 14 days of incubation at 28° C. The color schemeused was the Color Harmony Manual Fourth edition, 1958 (Container Corp.of America).

                                      Table 1                                     __________________________________________________________________________    Cultural Characteristics of Streptomyces sp. X 14547                          Agar   Amount of Growth     Color of Reverse                                  Medium Degree of Sporulation                                                                    Spore Mass Color                                                                        Substrate Mycelium                                __________________________________________________________________________    Yeast Malt                                                                           moderate to abundant                                                                     3 fe (silver gray)                                                                      2 nl (covert brown) at                            Extract                                                                              growth; well                                                                             mostly; some tufts                                                                      center; 2 ca (light                               (ISP-2)                                                                              sporulated; slightly                                                                     of a (white)                                                                            ivory) at edge                                           hygroscopic                                                            Oatmeal                                                                              abundant growth; well                                                                    3 fe (silver gray)                                                                      3 fe (silver gray) at                             (ISP-3)                                                                              sporulated           center and 2 dc                                                               (natural) at edge                                 Inorganic                                                                            abundant growth; well                                                                    3 fe (silver gray);                                                                     2 dc (natural)                                    Salts Starch                                                                         sporulated with edges and                                              (ISP-4)           flecks of b                                                                   (oyster white)                                              Glycerol                                                                             moderate growth;                                                                         2 fe (covert gray)                                                                      2 ec (bisquit) mostly;                            Asparagine                                                                           moderately to well                                                                       with patches and                                                                        2 ge (covert tan) at                              (ISP-5)                                                                              sporulated; slightly                                                                     edge of b edge                                                     hygroscopic                                                                              (oyster white)                                              Peptone                                                                              moderate growth; no                                                                      i (gray) where                                                                          i (gray) where not                                Yeast  sporulation; dark                                                                        not sporulated                                                                          sporulated                                        Extract                                                                              brown soluble                                                          Iron   pigment                                                                (ISP-6)                                                                       Tyrosine                                                                             poor growth; some                                                                        2 dc (natural)                                                                          3 li (beaver)                                     (ISP-7)                                                                              sporulation; slight                                                           amount of brown                                                               soluble pigment                                                        Czapek-Dox                                                                           poor growth; sparsely                                                                    b (oyster white)                                                                        b (oyster white)                                         sporulated                                                             Bennett's                                                                            moderate growth; well                                                                    2 fe (covert gray)                                                                      3 lg (adobe brown)                                       sporulated; hygro-                                                            scopic                                                                 Sabouraud                                                                            moderate growth; no                                                                      3 ie (camel) where                                                                      3 ie (camel) where                                Dextrose                                                                             sporulation                                                                              not sporulated                                                                          not sporulated                                    Thermo-                                                                              abundant growth; well                                                                    3 fe (silver gray)                                                                      3 pn (dark brown) at                              actinomyces                                                                          sporulated; hygro-                                                                       mostly; with tufts                                                                      center; and 3 ng                                  Fermenta-                                                                            scopic     of b (oyster                                                                            (yellow maple) at                                 tion              white)    edge                                              ATCC med-                                                                            moderate growth;                                                                         3 fe (silver gray);                                                                     3 pl (mustard brown)                              ium 5  well sporulated;                                                                         also areas of 3 dc                                          (American                                                                            groscopic  (natural)                                                   Type Culture                                                                  Collection                                                                    Catalogue of                                                                  Strains.                                                                      12th Ed.                                                                      1976. Rock-                                                                   ville, Md.)                                                                   Amidex abundant growth;                                                                         3 fe (silver gray)                                                                      3 pl (mustard brown)                                     well sporulated;                                                                         mostly; edges of b                                                                      at center; 2 cb (ivory                                   slightly hygro-                                                                          (oyster white)                                                                          tint) around edge                                        scopic                                                                 Starch abundant growth;                                                                         3 fe (silver gray);                                                                     2 dc (natural)                                    Casein well sporulated;                                                                         b (oyster white)                                                   hygroscopic                                                                              in one area                                                 __________________________________________________________________________

Table 2 below sets forth the morphological and physiologicalcharacteristics of Streptomyces sp. X-14547.

                  Table 2                                                         ______________________________________                                        Morphological and Physiological                                               Characteristics of Streptomyces sp. X-14547                                   Test              Response of Culture X-14547                                 ______________________________________                                        Chromagenic reaction,                                                                           +                                                           ISP-6                                                                         Melanin, ISP-7    + weak                                                      Spore surface     smooth                                                      Color of spore mass                                                                             gray                                                        Spore chain form  rectus-flexibilis                                           D-Glucose utilization                                                                           ++                                                          D-Xylose utilization                                                                            ++ to +                                                     L-Arabinose utilization                                                                         ++                                                          L-Rhamnose utilization                                                                          ++                                                          D-Fructose utilization                                                                          ++                                                          D-Galactose utilization                                                                         ++                                                          Raffinose utilization                                                                           -                                                           D-Mannitol utilization                                                                          ++                                                          i-Inositol utilization                                                                          ++                                                          Salicin utilization                                                                             -                                                           Sucrose utilization                                                                             -                                                           Cellulose utilization                                                                           -                                                           Reverse side pigment                                                                            -                                                           Soluble pigment   --Streptomycin sensitivity,                                 10 μg disc     +                                                           Nitrate reduction -                                                           Casein hydrolysis +                                                           Gelatin hydrolysis                                                                              +                                                           Starch hydrolysis +                                                           ISP-1 darkening   +                                                           NaCl (%) tolerance                                                                              5                                                           Temperature growth range °C.                                                             10- 37                                                      DAP isomer        Other than the MESO                                                           isomer                                                      ______________________________________                                         ++ = strong positive response; - = negative response                     

According to R. E. Buchanan and N. E. Gibbons, "Bergey's Manual ofDeterminative Bacteriology, 8th edition, 1974, Williams and Wilkins Co.,Baltimore, Md., culture X-14547 is similar to Streptomyces antibioticsbut when the two were compared there were differences noted inutilization of L-arabinose, melanin production on ISP-7, and reductionof nitrate.

The species Streptomyces X-14547 described herein includes all strainsof Streptomyces which form a compound of the Formula I and which cannotbe definitely differentiated from the culture number X-14547 and itssubcultures including mutants and variants. The compound of the FormulaI is identified herein and after this identification is known, it iseasy to differentiate the strains producing a compound of the Formula Ifrom others.

Streptomyces sp. X-14547, when grown under suitable conditions, producesa compound of the Formula I. A fermentation broth containingStreptomyces sp. X-14547 is prepared by inoculating spores or mycelia ofthe organism producing the compound of the Formula I into a suitablemedium and then cultivating under aerobic conditions. For the productionof a compound of the Formula I, cultivation on a solid medium ispossible but for production in large quantities, cultivation in a liquidmedium is preferable. The temperature of the cultivation may be variedover a wide range, 20°-35° C., within which the organism may grow but atemperature of 26°-30° C. and a substantially neutral pH are preferred.In the submerged aerobic fermentation of the organism for the productionof a compound of the Formula I, the medium may contain as the source forcarbon, a commercially available glyceride oil or a carbohydrate such asglycerol, glucose, maltose, lactose, dextrin, starch, etc. in pure orcrude states and as the source of nitrogen, an organic material such assoybean meal, distillers' solubles, peanut meal, cotton seed meal, meatextract, peptone, fish meal, yeast extract, corn steep liquor, etc. andwhen desired inorganic sources of nitrogen such as nitrates and ammoniumsalts and mineral salts such as ammonium sulfate, magnesium sulfate andthe like. It also may contain sodium chloride, potassium chloride,potassium phosphate and the like and buffering agents such as sodiumcitrate, calcium carbonate or phosphates and trace amounts of heavymetal salts. In aerated submerged culturing procedures, an anti-foamagent such as liquid paraffin, fatty oils or silicone compounds is used.More than one kind of carbon source, nitrogen source or anti-foam sourcemay be used for production of a compound of the Formula I.

The following Examples will serve to illustrate this invention withoutlimiting it thereto.

EXAMPLE 1 Tank fermentation of Streptomyces sp. X-14547

The antibiotic X-14547 producing culture is grown and maintained on anAmidex agar slant having the following composition (grams/literdistilled water):

    ______________________________________                                               Amidex   10.0                                                                 N-Z amine A                                                                            2.0                                                                  Beef extract                                                                           1.0                                                                  Yeast extract                                                                          1.0                                                                  CoCl.sub.2 . 6H.sub.2 O                                                                0.02                                                                 Agar     20.0                                                          ______________________________________                                    

The slant is inoculated with antibiotic X-14547 producing culture andincubated at 28° C. for 7-10 days. A chunk of the agar containing sporesand mycelia from the sporulated culture slant is then used to inoculatea 6-liter Erlenmeyer flask containing 2 liters of sterilized inoculummedium having the following composition (grams/liter distilled water):

    ______________________________________                                        Tomato pomace solids                                                                             5.0                                                        Distiller's dried solubles                                                                       5.0                                                        OM peptone         5.0                                                        Debittered yeast   5.0                                                        Corn starch        20.0                                                       CaCO.sub.3         1.0                                                        K.sub.2 HPO.sub.4 (anhydrous)                                                                    1.0                                                        ______________________________________                                    

Adjust pH to 7 with NaOH before autoclaving at 15-20 pound pressure for45 minutes.

The inoculated inoculum medium is incubated at 28° C. for 72 hours on arotary shaker, operating at 250 rpm with a 2-inch stroke.

A four liter portion of the resulting culture is then used to inoculate60 gallons in a 100 gallon fermentor having the following composition(grams/liter distilled water):

    ______________________________________                                               Glucose   10.0                                                                Edible molasses                                                                         20.0                                                                HySoy T    5.0                                                                CaCO.sub.3                                                                               2.0                                                         ______________________________________                                    

Adjust pH to 7.2 with NaOH before sterilization for 11/4 hours with 60lb./in² steam.

The inoculated medium is aerated with sterilized compressed air at arate of 3 cubic feet per minute and is stirred with agitators at 280rpm. The fermentation is carried out at 28° C. for 4-6 days.

EXAMPLE 2 Isolation of antibiotic X-14547 and co-metabolites3-ethyl-1,3-dihydro-3-methoxy-2H-indole-2-one and pyrrole-2-carboxylicacid

Step A. To the whole broth from a 100 gallon (380 liters) fermentationas set forth in Example 1 was added, after 4-6 days of growth, an equalvolume of ethyl acetate. After stirring for one hour the solvent layerwas separated and concentrated to 2 liters under reduced pressure. Theconcentrated solvent extract was washed with equal volumes of 1N HClthree times. The solvent was dried over anhydrous Na₂ SO₄ andconcentrated to an oil under reduced pressure. The oil was dissolved indiethyl ether and crude pyrrole-2-carboxylic acid crystals wereseparated by filtration. Recrystallization from ethanol/ether yieldedthe analytical sample of the above compound: mp 202°-203° C.

microanalysis: calcd for C₅ H₅ NO₂ (111.10): calcd %C, 54.06; %H; 4.54;%N, 12.60. found %C, 54.33; %H, 4.65; %N, 12.60.

Step B. The mother liquor was concentrated to an oil under reducedpressure, redissolved in 250 ml of acetonitrile and washed twice withequal volumes of n-hexane. The hexane washes were pooled and extractedwith 1/2 volume of methanol. The methanol extract was pooled with theacetonitrile and the solvent removed under reduced pressure. The oilysolid was dissolved in acetonitrile and after cooling to approximately3° C. overnight crystalline antibiotic X-14547 was recovered uponfiltration as a hemihydrate, mp 137° C., [α]_(D) -285° (C, 1 in CHCl₃).

microanalysis: calcd for C₃₁ H₄₃ NO₄.(H₂ O)₀.5 (502.70): %C, 74.07; %H,8.82; %N, 2.78; %O; 14.32. found: %C, 74.36; %H, 8.93; %N, 2.50; %O,13.81.

Step C. The CH₃ CN mother liquor was concentrated to an oily solid andsubjected to chromatography on a hexane slurry packed 600 g silica gel(Davison grade 62) column. The column was eluted with 250 ml of hexaneand then a gradient between 1 liter of 2% ethyl acetate in hexane to 1liter of ethyl acetate/hexane (3:1) and then 500 ml of ethyl acetate.Fractions of 6 ml each were collected and from fraction numbers 100 to200 subsequent to the solvent being removed under reduced pressure,additional antibiotic X-14547 was recovered. From fractions 201 to 290after concentration and crystallization from acetonitrile,3-ethyl-1,3-dihydro-3-methoxy-2H-indole-2-one was recovered. mp 179°

microanalysis calcd: for C₁₁ H₁₃ NO₂ (191.23): calcd: %C, 69.09; %H,6.85; %N, 7.33. found: %C, 69.02; %H, 6.96; %N, 7.19.

EXAMPLE 3 Tank fermentation of antibiotic X-14547

The antibiotic X-14547 producing culture is grown and maintained on anAmidax agar slant as described in Example 1 or on a starch-casein agarslant having the following composition (grams/liter distilled water):

    ______________________________________                                        Soluble starch     10.0                                                       Casein             1.0                                                        K.sub.2 HPO.sub.4 (anhydrous)                                                                    0.5                                                        MgSO.sub.4 (anhydrous)                                                                           0.5                                                        Agar               20.0                                                       ______________________________________                                    

Adjust pH to 7.4 with NaOH before autoclaving at 15-20 p.s.i. for twentyminutes.

The slant is inoculated with antibiotic X-14547 producing culture(Streptomyces sp. X-14547) and incubated at 28° C. for 7-10 days. Achunk of agar from the sporulated culture is then used to preparevegetative inoculum by inoculating a 6-liter Erlenmeyer flask containing2 liters of sterilized inoculum medium having the following composition(grams/liter distilled water):

    ______________________________________                                        Tomato pomace solids                                                                             5.0                                                        Distiller's dried solubles                                                                       5.0                                                        OM peptone         5.0                                                        Debittered yeast   5.0                                                        Corn starch        20.0                                                       CaCO.sub.3         1.0                                                        K.sub.2 HPO.sub.4 (anhydrous)                                                                    1.0                                                        ______________________________________                                    

pH is adjusted to 7.0 before autoclaving at 15-20 p.s.i. for forty-fiveminutes.

The inoculated inoculum medium is incubated for 72 hours at 28° C. on arotary shaker operating at 250 rpm with a 2-inch stroke.

Four liters of this culture are used to inoculate 60 gallons of thefollowing medium in a 100 gallon fermentor (grams/liter tap water):

    ______________________________________                                        Tomato pomace solids                                                                             5.0                                                        Distiller's dried solubles                                                                       5.0                                                        OM peptone         5.0                                                        Debittered yeast   5.0                                                        Corn starch        20.0                                                       CaCO.sub.3         1.0                                                        K.sub.2 HPO.sub.4 (anhydrous)                                                                    1.0                                                        Sag 4130 Antifoam  0.1                                                        (Union carbide)                                                               ______________________________________                                    

The pH of the medium is adjusted to 7.0 with NaOH before sterilizationfor 11/4 hours with 60 p.s.i. steam.

The inoculated medium is aerated with compressed air at a rate of 3cubic feet per minute and is stirred with agitators at 280 rpm. Thefermentation is carried out at 28° C. for 43 hours.

Five gallons of this culture are used to inoculate 350 gallons of thefollowing medium in a 1000 gallon tank utilizing the following medium(grams/liter tap water):

    ______________________________________                                        Tomato pomace solids                                                                             5.0                                                        Distiller's dried solubles                                                                       5.0                                                        OM peptone         5.0                                                        Dibittered yeast   5.0                                                        Corn starch        20.0                                                       CaCO.sub.3         1.0                                                        K.sub.2 HPO.sub.4 (anhydrous)                                                                    1.0                                                        Sag 4130 Antifoam  0.1                                                        (Union carbide)                                                               ______________________________________                                    

The pH of the medium is adjusted to 7.0 with NaOH before sterilizationfor 11/4 hours with 60 p.s.i.² steam.

The inoculated medium is aerated with compressed air at a rate of 3cubic feet per minute and is stirred with agitators at 280 rpm. Thefermentation is carried out at 28° C. for 118 hours.

Isolation of antibiotic X-14547 andco-metabolites-3-ethyl-1,3-dihydro-3-methoxy-2H-indole-2-one andpyrrole-2-carboxylic acid

Step A. To the whole broth from a 350 gallon (1350 liters) fermentationas set forth in Example 2 was added, after 118 hours of growth, an equalvolume of ethyl acetate. After stirring for one hour the solvent layerwas separated and concentrated to 7.25 liters under reduced pressure.The concentrated solvent extract was washed with 3 liters of 1 N HClthree times. The solvent was dried over anhydrous Na₂ SO₄ andconcentrated to an oil under reduced pressure. The oil was dissolved indiethyl ether and crude pyrrole-2-carboxylic acid crystals wereseparated by filtration. Recrystallization from ethanol/ether yieldedthe analytical sample of the above compound: mp 202°-203° C.

Microanalysis: calcd for C₅ H₅ NO₂ (111.10): calcd %C, 54.06; %H, 4.54;%N, 12.60. found %C, 54.33; %H, 4.65; %N, 12.60.

Step B. The mother liquor was concentrated to an oil under reducedpressure, redissolved in 1 liter of acetonitrile and washed twice withequal volumes of n-hexane. The hexane washes were pooled and extractedwith 1/2 volume of methanol. The methanol extract was pooled with theacetonitrile and the solvent removed under reduced pressure. The oilysolid was dissolved in acetonitrile and after cooling to approximately3° C. overnight crystalline antibiotic X-14547 was recovered uponfiltration as a hemihydrate, mp 137° C., [α]_(D) -285° (C, 1 in CHCl₃).

Microanalysis: calcd for C₃₁ H₄₃ NO₄.(H₂ 0)₀.5 (502.70): %C, 74.07; %H,8.82; %N, 2.78; %O; 14.32. found %C, 74.36; %H, 8.93; %N, 2.50; %O,13.81.

Step C. The CH₃ CN mother liquor was concentrated to an oily solid andsubjected to chromotography on a hexane slurry packed 600 g silica gel(Davison grade 62) column. The column was eluted with 1 liter of hexaneand then a gradient between 4 liters of 2% ethyl acetate in hexane to 4liters of ethyl acetate/hexane (3:1) and then 2 liters of ethyl acetate.Fractions of twenty five ml each were collected and from fractionnumbers 100 to 200 subsequent to the solvent being removed under reducedpressure, additional antibiotic X-14547 was recovered. From fractions201 to 290 after concentration and crystallization from acetonitrile,3-ethyl-1,3-dihydro-3-methoxy-2H-indole-2-one was recovered, mp 179°

Microanalysis calcd for C₁₁ H₁₃ NO₂ (191.23): calcd: %C, 69.09; %H,6.85; %N, 7.33. found: %C, 69.02; %H, 6.96; %N, 7.19.

EXAMPLE 5 Isolation of the sodium salt of antibiotic X-14547

The whole broth from another fermentation run was extracted with a onehalf volume of chloroform. The solvent layer was separated andconcentrated under reduced pressure to 2.35 liters, and washedsuccessively with equal volumes of 1 N HCl, 1 N NaOH and water. Thesolvent was dried over Na₂ SO₄, and concentrated to an oil. The oil wasredissolved in 2 liters of acetonitrile and washed with 2 liters ofn-hexane. The acetonitrile was concentrated to an oil and filteredthrough 970 grams of silica gel with 4 liters of methylene chloride andthen 8 liters of methylene chloride-acetone (1:1). The biologicallyactive fraction was chromatographed on a 300 gram slurry packed(methylene chloride) silica gel column and eluted with a gradientbetween 7 liters of methylene chloride and 7 liters of methylenechloride-diethyl ether-ethanol (48:48:14). Fractions numbered 290-460(twenty-five ml each) were pooled and concentrated to an oil. The oilwas dissolved in a small amount of acetonitrile and with the addition ofn-hexane the sodium salt of antibiotic X-14547 was recovered byfiltration as a white powder, containing 1 mole of hexane.

Calcd. C₃₁ H₄₂ N NaO₄.C₆ H₁₄ (599.83): %C, 74.09; %H, 9.08; %N, 2.34;%Na 3.83.

Found: %C, 74.00; %H, 8.86; %N, 2.10; %Na, 4.04.

EXAMPLE 6 Preparation of the thallium salt of antibiotic X-14547

A solution of 1.3 g of antibiotic X-14547 in ethyl acetate was firstwashed with 1NHCl, and then four times with an aqueous solution ofthallium hydroxide. The solvent was separated and concentrated to asmall volume under reduced pressure and after addition of CH₃ CN-C₂ H₅OH, crystalline thallium salt of antibiotic X-14547 was recovered, mp.194°-195° C.

Calcd. C₃₁ H₄₂ NO₄ Tl (697.05): %C, 53.42; %H, 6.07; %N, 2.01; %Tl29.32.

Found: %C 53.51; %H, 6.01; %N, 1.98; %Tl 28.96.

EXAMPLE 7 Preparation of the R-(+)-1-amino-1-(4-bromophenyl)-ethane saltof antibiotic X-14547

A solution of 493 mg (1 mmol) of antibiotic X-14547 in methylenechloride was added to a solution of 181 mg ofR-(+)-1-amino-1-(4-bromophenyl)-ethane in methylene chloride. Afteraddition of n-hexane and slow evaporation of solvent, the crystallinefinal product was recovered. Recrystallization from methylenechloride-hexane yielded crystals suitable for X-ray analysis, mp.128°-131°.*

Calcd. C₇₀ H₉₆ BrN₃ O₈ (1187.46): %C, 70.70; %H, 815; %N, 3.54; %Br,6.73.

Found: %C, 70.96; %H, 8.30; %N 3.68; %Br, 6.83.

Temperatures disclosed in this specification are in degrees celcius.

The following are various physical characteristics of antibioticX-14547:

The infrared absorption spectrum of antibiotic X-14547 in a KBR pelletis shown in FIG. 1. The antibiotic exhibits characteristics absorptionin the infrared region of the spectrum at the following wave lengthsexpressed in reciprocal centimeters:

Peaks occurred inter alia at 3140 (OH), 2740-2400 (carboxyl OH), 1735,1710 (C═O), 1650 (conjugated C═O) and 1627 cm⁻¹ (conjugated C═C).

Antibiotic X-14547 exhibits an oral toxicity (LD₅₀) in mice of 129 mg/kg(24 hours).

Antibiotic X-14547 has exhibited antimicrobial activity against avariety of gram-positive bacteria and mycobacterium as indicated inTable 3 below.

                  Table 3                                                         ______________________________________                                                               Antimicrobial                                                                 activity of                                                                   Antibiotic X-14547.                                               Culture     Minimum inhibitory                                                Collection No.                                                                            concentration* in                                      Name of Organism                                                                           ATCC     NRRL     Mcg/Ml                                         ______________________________________                                        Bacillus megaterium                                                                        8011              0.1                                            Sarcina lutea                                                                              9341              0.1                                            Bacillus species E                                                                         27859             0.2                                            Bacillus subtilis     558      0.1                                            Staphylococcus aureus                                                                      6538P             0.2                                            Bacillus species TA                                                                        27860             0.2                                            Mycobacterium phlei                                                                        355               3.1                                            Streptomyces cellulosae                                                                    3313              0.8                                            Paecilomyces varioti                                                                       26820             6.3                                            ______________________________________                                         *Lowest twofold dilution giving a zone of inhibition in an agar well          diffusion assay.                                                         

It has also been found and should be considered a part of the presentinvention that the novel 3-ethyl-1,3-dihydro-3-methoxy-2H-indole-2-oneco-metabolite also exhibits antimicrobial activity against a variety ofmicroorganisms as indicated in Table 4 below.

                  Table 4                                                         ______________________________________                                                      Culture Collection No.                                                                        M.I.C.*                                         Name of Organism                                                                            ATCC            in mcg/ml                                       ______________________________________                                        Escherichia coli                                                                            27856           5.0                                             Pseudomonas aeruginosa                                                                      8709            5.0                                             Klebsiella pneumoniae                                                                       27858           5.0                                             Staphylococcus aureus                                                                       6538P           5.0                                             Bacillus sp. TA                                                                             27860           2.5                                             Paecilomyces varioti                                                                        26820           2.5                                             ______________________________________                                         *Minimum inhibitory concentration per ml. in mcg.                        

As indicated above, antibiotic X-14547 and its salts together with itsco-metabolite 3-ethyl-1,3-dihydro-3-methoxy-2H-indole-2-one possessesthe property of adversely affecting the growth of certain gram-positivebacteria. They are ueful in wash solutions for sanitary purposes as inthe washing of hands and the cleaning of equipment, floors orfurnishings of contaminated rooms or laboratories.

Antibiotic X-14547 has also been found to improve ruminant feedutilization, i.e., improve the digestive efficiency of certainherbivorous animals, for example, cattle. A discussion of the mechanismwhereby feed is digested, degraded and metabolized in a ruminant animalcan be found in U.S. Pat. No. 3,839,557 issued Oct. 1, 1974 whichdiscloses the use of certain antibiotics in improving ruminant feedutilization and is incorporated herewith by reference.Economically-important ruminant animals are cattle, sheep and goats.

The effectiveness of antibiotic X-14547 in modifying the ratio ofvolatile fatty acids produced in the rumen (and thereby improve ruminantfeed utilization) is demonstrated by means of the following in vitrotesting.

Rumen fluid is obtained from a steer with a fistulated rumen. The steeris maintained on the following ration:

    ______________________________________                                        Corn                   89.93%                                                 Alfalfa meal           5.000%                                                 Soy bean oil meal      3.00%                                                  Limestone              0.80%                                                  NaCl                   0.60%                                                  ______________________________________                                    

    ______________________________________                                        Dicalcium phosphate     0.50%                                                 Trace minerals          0.025%                                                Vitamin premix additions                                                                              0.1%                                                  Vitamin A, TIU          4.0003                                                Vitamin D.sub.3, IU     0.801                                                 Vitamin E, TIU          3.002                                                 ______________________________________                                    

The rumen fluid is immediately strained through a #30 mesh sieve. Foreach fermentation, 75 ml of the resulting fluid is added to a 250 mlflask containing the following:

1 g of 80%:20% finely ground grain: hay ration;

1 ml of an 18% aqueous glucose solution (1 millimole per flask);

1.5 ml of a 3.1% aqueous urea solution (0.76 millimole per flask);

60 micromoles of each of the 10 essential amino acids (arginine,histidine, leucine, methionine, threonine, valine, lysine, isoleucine,phenylalanine, tryptophan);

1 ml of an aqueous solution of test drug to give either 10 or 25 μg/ml(calculated total volume of fermentation mixture of 80 ml); Each flaskis incubated at 38° C. in a shaking water bath equipped with a gassinghood. Carbon dioxide is continuously passed through the hood. After fourhours incubation, a 10 ml quantity of the fermentation fluid iscentrifuged at 14,000 rpm (approximately 30,000 xg) for 20 minutes in anInternational Centrifuge equipped with a No. 874 angle head. Three ml ofthe supernate is added to 1 ml of a 25% metaphosphoric acid solutioncontaining 23 micromoles 2-methyl valeric acid as an internal standard.The resulting fluid is permitted to sit at room temperature for 30minutes. The fluid is filtered through a 0.22 millimicron Milliporefilter and refrigerated until gas-liquid chromatographic analyses forvolatile fatty acids.

Gas-liquid chromatographic (GLC) analyses of four in vitro controlfermentations and two fermentations each with 10 and 25 ppm AntibioticX-14547 are set forth in the following table.

Ratios of moles of propionate (C₃) to acetate (C₂) plus n-butyrate (nC₄)in in vitro rumen fermentations

    ______________________________________                                                     μmoles C.sub.3 /μmoles C.sub.2 + μmoles nC.sub.4        Description    Replicate                                                      (Incubation Time)                                                                            fermentations                                                                              Means (± 1σ)                             ______________________________________                                        Controls (0 hrs.)                                                                            0.372                                                                         0.361                                                                         0.364                                                                         0.350        0.362 (± 0.009)                                Controls (4 hrs.)                                                                            0.393                                                                         0.337                                                                         0.427                                                                         0.388        0.386 (± 0.037)                                Antibiotic X-14547 (4 hrs.),                                                                 0.493        0.491                                             10 ppm         0.489                                                          Antibiotic X-14547 (4 hrs.)                                                                  0.531        0.521                                             25 ppm         0.510                                                          ______________________________________                                    

As shown in the above table the ratio of propionate (C₃) to acetates andn-butyrates is significantly improved. With the increase of propionatesrather than acetates from the carbohydrates, the efficiency ofcarbohydrate and therefore feed utilization is increased.

Administration of antibiotic X-14547 hereafter "Antibiotic" or"Antibiotic Compound" prevents and treats ketosis as well as improvesfeed utilization. The causative mechanism of ketosis is a deficientproduction of propionate compounds. A presently recommended treatment isadministration of propionic acid or feeds which preferentially producepropionates. It is obvious that encouraging propionate production fromordinary feeds will reduce incidence of ketosis.

It has been found that antibiotic X-14547 increases the efficiency offeed utilization in ruminant animals when it is administered orally tothe animals. The easiest way to administer the antibiotic is by mixingit in the animal's feed.

However, the antibiotic can be usefully administered in other ways. Forexample, it can be incorporated into tablets, drenches, boluses, orcapsules, and dosed to the animals. Formulation of the antibioticcompound in such dosage forms can be accomplished by means of methodswell known in the veterinary pharmaceutical art.

Capsules are readily produced by filling gelatin capsules with anydesired form of the desired antibiotic. If desired, the antibiotic canbe diluted with an inert powdered diluent, such as a sugar, starch, orpurified crystalline cellulose in order to increase its volume forconvenience in filling capsules.

Tablets of the antibiotics are made by conventional pharmaceuticalprocesses. Manufacture of tablets is a well-known and highly advancedart. In addition to the active ingredient, a tablet usually contains abase, a disintegrator, an absorbent, a binder, and a lubricant. Typicalbases include lactose, fine icing sugar, sodium chloride, starch andmannitol. Starch is also a good disintegrator as is alginic acid.Surface-active agents such as sodium lauryl sulfate and dioctyl sodiumsulphosuccinate are also sometimes used. Commonly-used absorbents againinclude starch and lactose while magnesium carbonate is also useful foroily substances. Frequently-used binders are gelatin, gums, starch,dextrin and various cellulose derivatives. Among the commonly usedlubricants are magnesium stearate, talc, paraffin wax, various metallicsoaps, and polyethylene glycol.

The administration of the antibiotic compound may be as a slow-pay-outbolus. Such boluses are made as tablets except that a means to delay thedissolution of the antibiotic is provided. Boluses are made to releasefor lengthy periods. The slow dissolution is assisted by choosing ahighly water-insoluble form of the antibiotic. A substance such as ironfiling is added to raise the density of the bolus and keep it static onthe bottom of the rumen.

Dissolution of the antibiotic is delayed by use of a matrix of insolublematerials in which the drug is inbedded. For example, substances such asvegetable waxes, purified mineral waxes, and water-insoluble polymericmaterials are useful.

Drenches of the antibiotic are prepared most easily by choosing awater-soluble form of the antibiotic. If an insoluble form is desiredfor some reason, a suspension may be made. Alternatively, a drench maybe formulated as a solution in a physiologically acceptable solvent suchas a polyethylene glycol.

Suspensions of insoluble forms of the antibiotic can be prepared innonsolvents such as vegetable oils such as peanut, corn, or sesame oil,in a glycol such as propylene glycol or a polyethylene glycol; or inwater, depending on the form of the antibiotic chosen.

Suitable physiologically acceptable adjuvants are necessary in order tokeep the antibiotic suspended. The adjuvants can be chosen from amongthe thickeners, such as carboxymethylcellulose, polyvinylpyrrolidone,gelatin, and the alginates. Many classes of surfactants serve to suspendthe antibiotic. For example, lecithin, alkylphenol oxide adducts,naphthalenesulfonates, alkylbenzesulfonates, and the polyoxyethylenesorbitan esters are useful for making suspensions in liquid nonsolvents.

In addition many substances which effect the hydrophilicity, density,and surface tension of the liquid can assist in making suspensions inindividual cases. For example, silicone anti-foams, glycols, sorbitol,and sugars can be useful suspending agents.

The suspendable antibiotic may be offered to the grower as a suspension,or as a dry mixture of the antibiotic and adjuvants to be diluted beforeuse.

The antibiotic may also be administered in the drinking water of theruminants. Incorporation into drinking water is performed by adding awater-soluble or water-suspendable form of the antibiotic to the waterin the proper amount. Formulation of the antibiotic for addition todrinking water follows the same principles as formulation of drenches.

The most practical way to treat animals with the antibiotic compound isby the formulation of the compound into the feed supply. Any type offeed may be medicated with the antibiotic compounds, including commondry feeds, liquid feeds, and pelleted feeds.

The methods of formulating drugs into animal feeds are well known. It isusual to make a concentrated drug premix as a raw material for medicatedfeeds. For example, typical drug premixes may contain from about one toabout 400 grams of drug per pound of premix. The wide range results fromthe wide range of concentration of drug which may be desired in thefinal feed. Premixes may be either liquid or solid.

The formulation of ruminant feeds containing the proper amounts ofantibiotic for useful treatment is well understood. It is necessary onlyto calculate the amount of compound which it is desired to administer toeach animal, to take into account the amount of feed per day which theanimal eats and the concentration of antibiotic compound in the premixto be used, and calculate the proper concentration of antibioticcompound, or of premix, in the feed.

All of the methods of formulating, mixing and pelleting feeds which arenormally used in the ruminant feed art are entirely appropriate formanufacturing feeds containing the antibiotic compound.

As has been shown, oral administration of the antibiotic beneficiallyalters the production of propionates relative to the production ofacetates in the rumen. It may therefore be postulated that the sametreatment would also benefit monogastric animals which ferment fibrousvegetable matter in the cecum since it would be expected that abeneficial change in the propionate/acetate ratio would occur upon oraladministration of the instant antibiotic. Horses, swine and rabbits areexemplary animals which digest a part of their food by cecalfermentation.

Antibiotic X-14547 also exhibits activity in the treatment ofhypertension in warm-blooded animals.

Antihypertensive activity is tested for in the DOCA-Na Sprague Dawley(Charles River) male rats weighing 170-210 grams. DOCA-Na hypertensionis produced by unilateral nephrectomy followed by subcutaneousimplantation of a 25 mg. desoxycortico sterone (DOCA) pellet. Animalsare placed in individual cages receiving 0.9% sodium chloride solutionand rat chow diet ad libitum. Two weeks are allowed to elapse from thetime of surgery for development of hypertension, i.e., systolic bloodpressure of at least 150 mm. Hg.

Systolic blood pressure and heart rate are measured indirectly from thetail of unanesthetized rats, using a pneumatic pulse transducer. Controlreadings are taken prior to drug and at 1, 3, 6 and 24 hours post drug.

The results are expressed as absolute values and percent changes fromcontrols.

                                      Table 4                                     __________________________________________________________________________    SYSTOLIC BLOOD PRESSURE (mmHg)                                                        DAY RAT                                                                              RAT                                                                              RAT                                                                              RAT                                                                              RAT                                                                              RAT     PCT                                                    1  2  3  4  5  6  MEAN CHANGE                                     __________________________________________________________________________    PRE DRUG                                                                      CONTROLS    223                                                                              203                                                                              203                                                                              199                                                                              203                                                                              209                                                                              206.7                                                                10 MG/KG × 3 days PO                               POST DRUG                                                                             1   216                                                                              178                                                                              184                                                                              198                                                                              151                                                                              203                                                                              188.3                                                                              -9.0                                       VALUES IN                                                                     (mmHg)  2   211                                                                              201                                                                              176                                                                              168                                                                              190                                                                              185.7                                                                            -10.3                                                   2   184                                                                              174                                                                              161                                                                              165                                                                              165                                                                              179                                                                              171.3                                                                              -17.1                                              3   214                                                                              195                                                                              193                                                                              219                                                                              198                                                                              211                                                                              205.0                                                                              -0.7                                               3   210                                                                              177                                                                              173                                                                              206                                                                              190                                                                              220                                                                              196.0                                                                              -5.2                                               4   197                                                                              172                                                                              229                                                                              212                                                                              179                                                                              228                                                                              202.8                                                                              -1.7                                       __________________________________________________________________________    CARDIAC RATE (BEATS/MIN)                                                      PRE DRUG                                                                      CONTROLS    440                                                                              360                                                                              460                                                                              410                                                                              400                                                                              400                                                                              411.7                                                                10 MG/KG × 3 days PO                               POST DRUG                                                                             1   480                                                                              360                                                                              450                                                                              440                                                                              390                                                                              400                                                                              420.0                                                                              2.0                                        VALUES IN                                                                     BEATS/MIN                                                                             2   430                                                                              320                                                                              360                                                                              490                                                                              370                                                                              430                                                                              400.0                                                                              -2.6                                               2   420                                                                              310                                                                              400                                                                              310                                                                              380                                                                              450                                                                              378.3                                                                              -8.1                                               3   380                                                                              360                                                                              360                                                                              360                                                                              370                                                                              350                                                                              363.3                                                                              -11.3                                              3   370                                                                              360                                                                              350                                                                              360                                                                              380                                                                              370                                                                              365.0                                                                              -10.8                                              4   370                                                                              320                                                                              360                                                                              350                                                                              340                                                                              400                                                                              356.7                                                                              -13.1                                      __________________________________________________________________________

For use as an anti-hypertensive agent, antibiotic X-14547 is formulated,using conventional inert pharmaceutical adjuvant materials, into dosageforms which are suitable for oral administration. Other dosage forms,e.g., parenteral, are possible but are not preferred. The oral dosageforms include tablets, capsules, dragees, suspensions, solutions and thelike. The identity of the inert adjuvant materials which are used informulating the active ingredients the active compounds into oral dosageforms will be immediately apparent to persons skilled in the art. Theseadjuvant materials, either inorganic or organic in nature, include, forexample, gelatin, albumin, lactose, starch, magnesium stearate,preservatives (stabilizers) melting agents, emulsifying agents, saltsfor altering osmotic pressure, buffers, etc., which can be incorporated,if desired, into such formulations.

Generally the drug is administered once or twice a day.

The quantity of antibiotic X-14547 which is present in any of the abovedescribed dosage forms generally varies from 1 to 10 mg. per unitdosage. The dosage administered to a particular patient is variable,based on the weight of the patient and the condition of the patient. Aneffective dosage amount of active agent can, therefore, only bedetermined by the clinician utilizing his best judgement on thepatient's behalf.

An example of a tablet formulation is as follows:

    ______________________________________                                        Tablet Formulation                                                                                  Per Tablet                                              ______________________________________                                        Antibiotic X-14547      1.0 mg.                                               Lactose Anhydrous       137.0 mg.                                             Corn Starch             20.0 mg.                                              Microcrystalline Cellulose Ph 101                                                                     40.0 mg.                                              Magnesium Stearate      2.0 mg.                                                                       200 mg.                                               ______________________________________                                    

Procedure:

1. The drug was premixed with a part of the lactose, in a suitable mixerand thereafter milled.

2. The mixture was further blended with the cornstarch the remaininglactose and the cellulose for 15 minutes and thereafter milled.

3. The mixture was thereafter blended with the magnesium stearate fortwo minutes.

4. The tablets were compressed at a tablet weight of 200 mg using tabletpunches having a diameter of approximately 1/4 inch. The tablets may beeither flat or biconnex and may be scored if desired.

1. A process for the production of the antibiotic X-14647 having thefollowing formula ##STR3## which comprises cultivating a strain ofStreptomyces sp. X-14547, NRRL 8167, in an aqueous carbohydrate solutioncontaining a nitrogenous nutrient under submerged aerobic conditions andthereafter isolating the X-14547 antibiotic from said solution.